The permeability of the neutral form of tertiary amine local anesthetics across squid axon membranes was studied by utilizing three different experimental methods: (1) permeability was measured by monitoring the time derivative of the squid action potential and the results were analyzed in terms of a diffusion reaction equation of local anesthetics to obtain their permeabilities; (2) the influx of local anesthetic into the axon was measured by use of the radioisotope tracer technique; and (3) the desorption rates of the neutral form of local anesthetics from lipid monolayers were measured and the desorption rate was correlated with permeability. The relative permeabilities obtained for procain, lidocain, and tetracain by the above three methods were in agreement. The order of relative permeabilities was procain greater than lidocain greater than tetracain.